Adverse outcome pathways (AOPs) provide a framework to organize and weigh the evidence linking a toxicant’s initial interactions with molecules in the cell to adverse outcomes of regulatory concern. AOPs are constructed in modules that include key events (KEs) and key event relationships (KERs). Quantitative understanding of the KERs is critical for the development of predictive toxicological models. The objective of this project was to investigate the ability to define the quantitative associations of the KERs upstream, and contained in, an existing AOP (#296): “Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations”. The data supporting quantitative associations between these KERs was gathered through literature review and experimental methods. I first used systematic literature review tools to develop and apply a pragmatic and transparent method to search the literature for AOP evidence. A broad search, covering all of the KERs of interest, was initially conducted. This search, which retrieved more than 230 thousand articles, demonstrates the data-rich nature of the AOP. An artificial intelligence informed prioritization of the top 100 articles were then examined in detail. This approach identified 39 articles containing qualitative empirical support for the AOP, but limited quantitative evidence of the KERs. A second search was conducted to address the need for quantitative evidence as well as the lack of evidence for the KER between and increase in reactive oxygen species (ROS) and oxidative DNA damage. The second search retrieved 12 articles that could be used to define a quantitative relationship between cellular ROS and oxidative DNA damage. To begin to address gaps in quantitative understanding, I then conducted experiments in the laboratory to measure oxidative DNA damage, DNA strand breaks, chromosomal aberrations, and mutations in TK6 cells after exposure to a range of concentrations of 4-Nitroquinoline 1-oxide (4NQO: a prototype ROS producing agent). An increase in both oxidative DNA damage and DNA strand breaks was observed after 2, 4, and 6 h exposures with the high throughput comet assay (CometChip). An increase in the incidence of micronuclei was observed after a 24 h exposure to a low concentration of 4NQO, as measured with the flow cytometry micronucleus assay, while high cytotoxicity was found at higher concentrations. Lastly an increase in mutation frequency was observed with Duplex Sequencing, an error-corrected sequencing technology. Additionally, an increase in the proportion of C>A transversions was observed, consistent with the expected mutations following oxidative DNA lesions. Overall, my work contributes to the quantitative understanding of AOP #296 and this project serves as a key example of AOP-informed study design, highlighting notable challenges in characterizing quantitative relationships.

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The G protein-coupled estrogen receptor GPER/GPER1, also known as GPR30, was originally cloned as an orphan receptor and later shown to be specifically activated by 17-ß-estradiol. This has led to its classification as an estrogen receptor and expanded the perspective on the mechanisms underlying the rapid estrogenic effects reported over the years. GPER is strongly expressed in the central nervous system and peripheral tissues and appears to be involved in a wide variety of physiological and pathological processes. Estrogens are known to alter the processing of nociceptive sensory information and analgesic responses in the central nervous system. Both analgesic and pro-nociceptive effects of estrogens have been reported. Some pro-algesic estrogenic responses have a short latency, suggesting a non-genomic mechanism of action. Immunohistochemical studies in rodents prove the existence of GPER in pain-relevant areas of the nervous system such as dorsal root ganglia, superficial dorsal horn of the spinal cord, periaqueductal gray (PAG), amygdala, trigeminal sensory nucleus and thalamus. In the periphery, activation of GPER results in pro-nociceptive effects. However, GPER involvement in pain processing at central levels is largely unexplored. Thus, the work presented in this thesis was aimed at investigating whether GPER modulates nociception at spinal and supraspinal sites. The behavioral response to GPER activation in the spinal cord and PAG was evaluated in an acute grooming test (scratching, biting and licking behavior) and in the hot plate test, respectively. Intrathecal challenge of mice with the GPER agonist G-1 (0.1-1 nmol) induced a dose-dependent increase in pain-related behaviors, that was abolished by pre-treatment with the GPER antagonist G15 (1-10 nmol), confirming GPER specificity of the response. Likewise, intra-PAG microinjection of G-1 (10-100 pmol) to rats reduced the nociceptive threshold in the hot plate test, an effect that was G15 sensitive. To obtain further insight on the mechanisms involved in the behavioral effects observed in whole animals, we tested the effect of GPER ligands on neuronal membrane potential, intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) accumulation. The membrane depolarization and the increases in [Ca2+]i and ROS levels are markers of neuronal activation, underlying pain sensitization in the spinal cord and pain facilitation in the PAG. Electrophysiological recordings from superficial dorsal horn and lateral PAG neurons indicate neuronal depolarization upon G-1 application, an effect that was fully prevented by G15 pre-treatment. Both cultured spinal neurons and cultured PAG neurons responded to G-1 administration by elevating [Ca2+]i and mitochondrial and cytosolic ROS levels. In the presence of G15, G-1 did not elicit the calcium and ROS responses. Collectively, these results demonstrate that GPER modulates both the ascending and descending pain pathways to increase nociception via cytosolic calcium elevation and ROS accumulation in spinal and PAG neurons, respectively. These findings broaden the current knowledge on GPER involvement in physiology and pathophysiology, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the mechanisms involved. Moreover, we show for the first time ROS accumulation downstream of GPER activation, extending the current understanding of GPER signaling. / Pharmacology

Bud dormancy is an essential characteristic of deciduous woody perennials, including apple, to cope with the low temperatures during winter. The release from dormancy and subsequent budburst in apple can only occur after fulfillment of chilling and heat requirements. In the Mid-Atlantic region, dormancy release and bud break of apple often coincide with late-spring freezes that cause severe damages to flowers, and small fruitlets. Therefore, the present study aimed to better understand mechanisms underlying bud dormancy in apple, with an ultimate goal of exploring chemical and/or genetic approaches for bloom-time modulation to avoid spring frost. Using two apple cultivars, 'Cripps Pink' and 'Honeycrisp,' representing early- and late-blooming cultivars, respectively, the present study specifically investigated the accumulation kinetics of plant hormones, carbohydrates, and reactive oxygen species (ROS) throughout the dormancy-regrowth cycle. Our results indicated that both cultivars required 1000 chilling hours for endodormancy release, but 'Honeycrisp' required 1000 growing degree hours (GDHs) more than 'Cripps Pink' for ecodormancy release and budburst. Among plant hormones, abscisic acid (ABA) showed remarkably elevated levels in the dormant buds of both cultivars during endodormancy, but its levels were significantly higher in 'Honeycrisp'. The decline of the ABA level at bud burst was combined with increased levels of cytokinin (CK). The ABA accumulation pattern during dormancy paralleled with an upregulation and downregulation of ABA biosynthetic and catabolic genes, respectively. On the other hand, the levels of hydrogen peroxide (H2O2) and superoxide (O2.-) were significantly higher in 'Cripps Pink' than 'Honeycrisp', particularly by the time of endodormancy and ecodormancy release, respectively. Our findings also showed a gradual decline in starch levels with the dormancy progression and increased levels of total soluble sugars (TSS) that were generally higher in the early-blooming cultivars. Transcriptomic profiling and module-trait relationship identified two modules that contrast between two cultivars mainly during eco-dormancy. Gene ontology (GO) analysis indicated that these DEGs were mostly involved in pathways related to hormones and signaling and co-expressed with H2O2 whereas, during ecodormancy pathways related to glutathione metabolism, auxin biosynthesis, carbohydrate metabolism and reproductive development were co-expressed with O2.-. Together, our results suggest that the contrasting bloom dates between 'Cripps Pink' and 'Honeycrisp' can be explained, at least partially, by the differential accumulation levels of ABA, ROS, antioxidants, and their associated genes in the buds of these cultivars throughout the dormancy cycle. / Doctor of Philosophy / Spring frosts represent a significant threat to apple production in many fruit-producing states of the United States including Virginia. The risk of frost damage is rising due to global climate change, and there is a high demand for effective measures to reduce frost damage. Exogenous applications of plant growth regulators (PGRs) to delay bloom has been suggested as an effective frost avoidance strategy, but with limited success. Therefore, the present study aimed to investigate molecular and biochemical pathways regulating bud dormancy and bloom time in apple, which can ultimate lead to novel approaches for bloom delay and frost mitigation. To this end, the accumulation patterns of major plant hormones (e.g. abscisic acid, ABA, cytokinin, CK and jasmonic acid, JA), reactive oxygen species (ROS) and carbohydrates (e.g. starch, sucrose, glucose and fructose) were thoroughly monitored throughout the dormancy-regrowth cycle in two apple cultivars, 'Cripps Pink' and 'Honeycrisp,' representing early- and late-blooming cultivars, respectively. Both these cultivars had similar chilling requirements (1000 chilling hours) but differed in their heat requirements; with 'Honeycrisp' requiring 1000 growing degree hours more than 'Cripps Pink'. Among plant hormones, ABA increased with the progression of dormancy and decreased with dormancy release in both cultivars. However, ABA levels were significantly higher in 'Honeycrisp' compared to 'Cripps Pink'. On the contrary, during dormancy release, the growth-promoting hormone, CK, increased earlier in 'Cripps Pink'. The levels of ROS, e.g., hydrogen peroxide (H2O2), and superoxide (O2.-), were also higher in 'Cripps Pink' than 'Honeycrisp', particularly by the time of endodormancy and ecodormancy release, respectively. Our data showed that starch levels generally declined during dormancy, whereas soluble sugars increased. However, there was no significant alternations in the carbohydrate accumulation profiles between the two cultivars that could account for the differences in their bloom dates. These results were verified further at the transcriptomic level. Using the RNA-sequencing technology, identified two modules that contrast between two cultivars mainly during eco-dormancy. Gene ontology (GO) analysis indicated that these genes were mostly involved in pathways related to hormones and signaling and co-expressed with H2O2 whereas during ecodormancy pathways related to glutathione metabolism, auxin biosynthesis, carbohydrate metabolism and reproductive development were co-expressed with O2.-. Overall, our results suggest that ABA, cytokinin, H2O2, and O2.- may, at least partially, explain the differences in the bloom time between the two apple cultivars. Further analysis of these molecules and their associated genes in other apple cultivars with contrasting bloom dates is necessary for better understanding of bloom time regulation in apple and developing strategies against frost damage.

Deficits in skeletal muscle mitochondrial metabolism are associated with a wide variety of chronic skeletal muscle and metabolic-related diseases, including diabetes and sarcopenia. Even in patients with advanced skeletal muscle-related diseases, exercise is a well-established method to improve skeletal muscle mitochondrial metabolism, culminating in enhanced whole-body metabolism and decreased disease severity. In response to exercise, there is an increase in reactive oxygen species (ROS) production. Historically, ROS were solely considered to drive disease development. However, ROS are also required for physiological adaptation and many questions still remain regarding their downstream pathways. One significant producer of skeletal muscle ROS with exercise is Nadph oxidase 4 (Nox4). Nox4 is unique compared to other Nox members as it predominantly produces hydrogen peroxide (H2O2), an effective signaling molecule. Here we demonstrate an essential role for Nox4 in mediating the beneficial effects of exercise. This work will contribute to our understanding of physiological ROS and their downstream targets by identifying a novel role for Nox4 in exercise adaptation. Further defining the molecular events that promote exercise adaptation will be essential for formulating new treatment strategies for patients with chronic metabolic diseases. / Doctor of Philosophy / Exercise is a widely effective tool for both preventing and reversing disease. Even patients with advanced skeletal muscle and metabolic-related diseases can benefit from continual and repeated exercise training. While decades of work have supported the effectiveness of exercise as a therapeutic intervention, the mechanistic understanding of what occurs at the cellular level remains incomplete. Here, we elucidate a novel pathway mediating important metabolic adaptations to exercise. In response to exercise stress, reactive oxygen species (ROS) are produced in skeletal muscle. ROS facilitate metabolic adaptations to meet the body's need for increased energy. One significant source of ROS comes from Nadph oxidase 4 (Nox4) which plays an essential role in metabolic regulation. The skeletal muscle metabolic response to stress is largely dependent on adaptations that include changes in gene expression, substrate oxidation, and mitochondrial metabolic adaptations. These mitochondrial adaptations include mitochondrial recycling after exercise in skeletal muscle (referred to as mitophagy). We have shown that Nox4 increases the expression of a subset of metabolic genes, is required for substrate oxidation after exercise, and is important for exercise-induced mitophagy.